N-type, CaV2.2 calcium channels are critically important proteins that regulate release of glutamate and substance P from nociceptors in the superficial dorsal horn of the spinal cord. Nociceptors respond to harmful signals such as high heat and, when functioning normally, are highly protective. In certain chronic pain conditions, such as after peripheral nerve injury, normal heat and touch are perceived as painful, and ongoing spontaneous activity of pain circuits can cause unrelenting pain. Understanding the molecular and cellular changes that occur during the transition from normal to chronic pain states is the key to improving current - and inadequate - therapies to treat chronic pain. Presynaptic N-type calcium channels in the spinal dorsal horn are major targets of drugs, including morphine, that are used to treat neuropathic and chronic pain syndromes. In the first two phases of this project we discovered that distinct neuronal populations in the mammalian nervous system express different isoforms of N-type calcium channels. Most importantly, we discovered a novel N-type channel isoform in nociceptors, which we predicted is particularly sensitive to inhibition by morphine when neurons fire at high rates. This exciting discovery has raised the possibility that drugs or therapies might be developed that act selectively on the N-type channels in the nociceptors that are responsible for chronic pain with less influence on N-type channels elsewhere in the nervous system. In the third phase of our project we will i) identify genomic DNA and pre-mRNA mechanisms that control expression patterns of N-type calcium channel isoforms in nociceptors and how these mechanisms achieve cell-specificity, ii) identify the precise N-type calcium channel isoforms that function at presynaptic nerve terminals in the spinal dorsal horn and establish their unique responsiveness to opiates and other analgesics, and iii) show how the activity and properties of presynaptic N-type calcium channels are altered in chronic pain states. To complete this project we have generated several genetically modified strains of mice in which we remove individual splice options, reducing the number of N-type channel isoforms available. In addition we use optogenetics combined with retrolabeling to examine synaptic events in dorsal horn projection neurons that arise from light-induced activation of thermosensing afferents. We integrate analyses of gene regulation, ion channel function, synaptic transmission, behavior and pharmacology to advance our understanding of this highly important group of calcium ion channels. We aim to identify the precise calcium channel isoforms that control transmission of normal and abnormal pain signals from peripheral nociceptors to central processing sites in the brain. Our results should lead to new strategies to help millions of neuropathic and chronic pain sufferers.